A variety of assay techniques are available for testing a biological sample obtained from any of a variety of sources for the presence of a nucleic acid sequence that may indicate the presence, for example, of a particular bacterium, virus or other pathogen, including a particular strain or mutant. Assays are also available for testing such samples for the presence of a nucleic acid sequence of the subject's own genomic DNA that may indicate the presence, for example, of one or another disease-related genetic mutation. Assays may include oligonucleotide probes bearing detectable labels, for example, P32 or fluorophores. Nucleic acids, either DNA or RNA, in a sample may be probed directly. Alternatively, assays may include amplification of target sequences by any of several amplification techniques, for example, PCR, NASBA or TMA. Amplification assays may be monitored in real time utilizing intercalating dyes, for example SYBR green, or fluorescently labeled probes, such as 5′ nuclease probes Livak, K. J. et al. (1995), Oligonucleotides with fluorescent Dyes at Opposite Ends Provide a Quenched Probe System Useful for Detecting PCR Product and Nucleic Acid Hybridization, PCR Meth. Appl. 4: 357-362, dual FRET probes, Espy, M. J. et al. (2002), Detection of Vaccinia Virus, Herpes Simplex Virus, Varicella-Zoster Virus, and Bacillus anthracis by LightCycler Polymerase Chain Reaction after Autoclaving: Implications for Biosafety of Bioterrorism Agents, Mayo Clin. Proc. 77: 624-628, or molecular beacon probes, Tyagi, S. and Kramer, F. R. (1996), Molecular Beacons: Probes that Fluoresce upon Hybridization, Nature Biotechnol. 14: 303-308; Tyagi, S. et al. (1998), Multicolor Molecular Beacons for Allele Discrimination, Nature Biotechnol. 16: 49-53. Real-time multiplex assays utilizing PCR amplification have been demonstrated with TaqMan dual-labeled linear probes and the 5′ nuclease detection process and, alternatively, with PCR amplification and molecular beacon probes, Tyagi. S. et al. (1998), supra; Vet, J. A. et al. (1999), Multiplex Detection of Four Pathogenic Retroviruses Using Molecular Beacons, Proc. Natl. Acad. Sci. USA 96: 6394-6399; El-Hajj, H. et al. (2001), Detection of Rifampin Resistance in Mycobacterium tuberculosis in a Single Tube with Molecular Beacons, J. Clin. Microbiol. 39: 4131-4137. Fluorescence-based multiplex assays currently are limited to about eight targets per sample by the need to minimize overlaps in emission spectra of fluorophores and, hence, are not expandable for use as highly multiplexed screening assays.
Highly multiplexed assays rely on spatial segregation of targets for signal resolution. Spatial segregation enables the use of coding schemes involving combinations of differently colored fluorophores (combinatorial coding), combinations of different amounts of each fluorophore (ratio coding), and both. One example of an assay with spatial segregation is fluorescence in situ hybridization, or FISH, for chromosomal analysis. Speicher et al. (1996), Karyotyping Human Chromosomes by Combinatorial Multi-fluor FISH, Nature Genet. 12: 368-375, for example, report the use of 27 probes combinatorially labeled using a set of six different fluorophores for analysis of chromosome spreads. Similarly, segregation of transcription sites in cell nuclei has enabled combinatorial coding utilizing multiple, singly labeled probes per site, as well as ratio coding. Singer, R. H., International (PCT) patent application WO 00/65094; Levsky, J. M. et al. (2002), Single-Cell Gene Expression Profiling, Science 297: 836-840. Another spatial-segregation probe technique is the use of multiplex probe arrays, including arrays on DNA chips. Schena, M. et al. (1995), Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray, Science 20: 467-470; Gingeras, T. R. et al. (1998), Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic Mycobacterium DNA Arrays. Genet. Res. 8: 435-448; Han et al. (2001), Quantum-Dot-Tagged Microbeads for Multiplexed Optical Coding of Biomolecules, Nature Biotechnol. 19, 631-635. Another segregation approach is the use if electrophoresis to separate ligated probe pairs of differing lengths. Tong, A. K. et al. (2001), Combinatorial Fluorescence Energy Transfer Tags for Multiplex Biological Assays, Nature Biotechnol. 19: 756-759. In that method, a variation of the oligonucleotide ligation assay (“OLA”) for SNP detection, differently labeled probes are ligated to capture probes on a target, and the hybrids are immobilized, washed, released, separated from one another by electrophoresis, and read for each probes' fluorescent code, which is a combination of colors and ratios. Array methods and electrophoretic methods remain technically complex, requiring many separate steps, including amplification, hybridization, washing and analysis.
There is not currently available a homogeneous fluorescence hybridization assay that is suitable for use as a highly multiplexed screening assay, despite the need for such an assay. During suppressive treatment of the HIV-1 virus with protease inhibitors, for example, any of about 30 mutations is likely to proliferate over time and to require change in treatment. Hirsch, M. S. et al. (1998), Antiretroviral Drug Resistance testing in Adults with HIV Infection, JAMA 279: 1984-1991. Lacking a highly multiplexed screening assay, current practice is to sequence the virus in response to a patient's increase in viral load. Sequencing is made difficult by the fact that the arising mutant is not the only allele present. During initial diagnosis of a patient with particular symptoms, for example fever, there is available no highly multiplexed homogeneous assay to screen for an early indication of one of numerous possible infectious agents that may be the cause of the patient's symptoms.
An aspect of this invention is highly multiplexed homogeneous assays for screening samples for the presence of a target nucleic acid sequence from among at least ten, and as many as 60 or more, possible targets, utilizing conventional fluorescence detection equipment and techniques, and fluorescently labeled hybridization probes.
Another aspect of this invention is such screening assays that employ target amplification, optionally with real-time detection, that are capable of detecting small amounts of pathogens that may be found in otherwise sterile samples such as blood.
Yet another aspect of this invention is kits and oligonucleotide sets for carrying out particular screening assays according to this invention.